JoVE General
DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning
Department of Pharmacology, University of California, Davis
We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.
JoVE General
Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics
Department of Marine Sciences, University of Georgia (UGA)
We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.
JoVE General
Microarray Analysis for Saccharomyces cerevisiae
Vermont Genetics Network, The University of Vermont
In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.
JoVE General
Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato
1Plant Pathology and Plant-Microbe Biology, Cornell University, 2Boyce Thompson Institute for Plant Research
Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.
JoVE Neuroscience
Transcriptome Analysis of Single Cells
1Department of Pharmacology, University of Pennsylvania, 2The Penn Genome Frontiers Institute, University of Pennsylvania
In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.
JoVE General
In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection
RNA Biology, New England Biolabs
This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.
JoVE Neuroscience
Single-cell Profiling of Developing and Mature Retinal Neurons
Department of Genetics, Development and Cell Biology, Neuroscience Program, Iowa State University
A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.
JoVE General
Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides
School of Biology, Georgia Institute of Technology
This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.
JoVE Neuroscience
Selection of Aptamers for Amyloid β-Protein, the Causative Agent of Alzheimer's Disease
1Department of Neurology, David Geffen School of Medicine, 2Molecular Biology Institute, University of California, Los Angeles, 3Brain Research Institute, University of California, Los Angeles
Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease.
JoVE General
A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA
1Department of Molecular and Cellular Biology, Brown University, 2Center for Computational Molecular Biology, Brown University
Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.
JoVE General
Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 3
Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology
Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.
JoVE Immunology and Infection
Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery
1Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, 2Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, 3Shared Resource-DNA/RNA Peptide, Beckman Research Institute of City of Hope
Several 2’-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.
JoVE Neuroscience
Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain
Max Planck Institute of Psychiatry
A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.
JoVE General
Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
1Regenerative Biology, Morgridge Institute for Research, 2Department of Cell & Regenerative Biology, University of Wisconsin, 3Department of Molecular, Cellular, & Regenerative Biology, University of California
Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.
JoVE Immunology and Infection
Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays
1The virology Core at the HIV Drug Resistance Program, NCI-Frederick, 2Division of Infectious Diseases, University of Pittsburgh, 3Department of Molecular Biology and Microbiology, Tuffts University
Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.
JoVE General
Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2
Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology
Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.
JoVE General
Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1
Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology
Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.
JoVE General
Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq
Department of Biology, New Mexico State University
A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.
JoVE Immunology and Infection
TransFLP — A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination
Global Health Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL)
A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.
JoVE General
Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos
1Institut de Recherches Cliniques de Montréal (IRCM), 2Department of Biochemistry, Université de Montréal
Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.
JoVE General
DNA Methylation: Bisulphite Modification and Analysis
1Epigenetics Group, Cancer Research Program, Garvan Institute of Medical Research, 2St Vincent's Clinical School, University of NSW
The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.
JoVE General
iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution
1Laboratory of Molecular Biology, Medical Research Council - MRC, 2European Bioinformatics Institute, EMBL Heidelberg, 3Computer and Information Science, University of Ljubljana, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute
The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.
JoVE Clinical and Translational Medicine
Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR
1Laboratorio de Investigación y Desarrollo (LID), Universidad Peruana Cayetano Heredia, 2Bloomberg School of Public Health, Johns Hopkins University, 3Laboratorio de Diagnostico Molecular, Facultad de Medicina, University of Concepcion,Chile, 4University of California San Diego School of Medicine
A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children’s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.
JoVE General
RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs
Department of Biological Sciences, Union College
By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.
JoVE General
Expression Analysis of Mammalian Linker-histone Subtypes
We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.
JoVE Bioengineering
Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming
1Florey Neuroscience Institutes and Centre for Neuroscience, University of Melbourne, 2Fluid Dynamics Group, CSIRO Materials Science and Engineering, 3Swinburne University of Technology, Faculty of Engineering and Industrial Sciences
We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.
JoVE General
Processing the Loblolly Pine PtGen2 cDNA Microarray
1Warnell School of Forestry and Natural Resources, University of Georgia (UGA), 2Instituto de Biologia Experimental e Tecnológica, Instituto Tecnologia Química e Biológica UNL, Av. da República
The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.
JoVE General
Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
1Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 2Deparment of Biochemistry, Research and Innovation Centre, University of Regina, 3Department of Medical Genetics and Microbiology, University of Toronto
Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.
JoVE Immunology and Infection
Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs
1Plant Sciences Institute, Agricultural Research Service, United States Department of Agriculture, 2Molecular Plant Pathology Laboratory, Agricultural Research Service, United States Department of Agriculture
A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.
JoVE Immunology and Infection
Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy
Department of Pathology, University of Texas Medical Branch
The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.
JoVE General
Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting
1School of Life Sciences, University of Warwick, 2Warwick Systems Biology, University of Warwick
A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.
JoVE General
Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue
1Department of Structural and Molecular Neuroscience, McLean Hospital, 2Department of Psychiatry, Harvard Medical School, 3Department of Psychiatry, Beth Israel Deaconess Medical Center
We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.
JoVE Immunology and Infection
Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field
1USGS Western Ecological Research Center, 2Wildlife Health Center, University of California, Davis, 3Department of Population Health and Reproduction, University of California, Davis, 4Department of Veterinary and Biomedical Sciences, University of Minnesota, 5Science Applications International Corporation
This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.
JoVE General
A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica
1Department of Biological Sciences, The University of Alabama, Huntsville, 2USDA-APHIS-PPQ, Center for Plant Health Science and Technology
We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).
JoVE Immunology and Infection
Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3
Laboratory Program, BC Centre for Excellence in HIV/AIDS
HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.
JoVE General
Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library
The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.
JoVE Immunology and Infection
Avian Influenza Surveillance with FTA Cards: Field Methods, Biosafety, and Transportation Issues Solved
1Resource Ecology Group, Wageningen University, 2Section for Zoonotic Ecology and Epidemiology, School of Natural Sciences, Linnaeus University, 3Centre for Wildlife Ecology, Simon Fraser University
A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.
JoVE General
Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay
Thermo Scientific Solaris qPCR Products
The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.
JoVE Neuroscience
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
1Department of Neurology and Committee on Neurobiology, The University of Chicago Medical Center, 2Department of Neurology, The University of Chicago Medical Center
Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.
JoVE General
In Situ Hybridization for the Precise Localization of Transcripts in Plants
The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.
JoVE General
Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence
1Department of Medicine, Albert Einstein College of Medicine, 2Diabetes Research and Training Center, Albert Einstein College of Medicine, 3Department of Genetics, Albert Einstein College of Medicine
An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.
JoVE Immunology and Infection
Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens
1Department of Laboratory Medicine, University of California, San Francisco, 2Division of Infectious Diseases, University of California, San Francisco
The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.
JoVE Immunology and Infection
Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)
Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.
JoVE Bioengineering
High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
1The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, 2Center for Reproductive Sciences, University of California San Francisco, 3Department of Urology, University of California San Francisco, 4Department of Cell and Tissue Biology, University of California San Francisco, 5Fluidigm Corporation, Fluidigm Corporation, 6Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Center, 7UCSF - Helen Diller Family Comprehensive Cancer Center, University of California San Francisco
Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.
JoVE General
Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm
Department of Biology, Saint Louis University
Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.
JoVE Immunology and Infection
Generation of Recombinant Influenza Virus from Plasmid DNA
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Microbiology and Medicine, and Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine
Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.
JoVE Immunology and Infection
Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors
Microbiology, Immunology, and Pathology, Colorado State University
Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.
JoVE General
MISSION esiRNA for RNAi Screening in Mammalian Cells
Max Planck Institute of Molecular Cell Biology and Genetics
Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.
JoVE General
Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)
1Centre for Medical Parasitology, Department of International Health, Immunology & Microbiology, Faculty of Health Sciences, University of Copenhagen, 2Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), 3Institute of Infection and Immunology Research, School of Biology, University of Edinburgh
Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.
JoVE Immunology and Infection
Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory
A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.
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